Easy-to-Follow Protocol For Western Blot Analysis & Immunofluorescence Staining

 

 Protocol for Western Blot for Detection of Protein of Interest

1. Cell Lysis. 

The total protein is extracted from cells and tissues using ice-cold cell lysis reagents such as T-PER Tissue Protein Extraction Reagent (Thermo Scientific, USA) supplemented with 1% Triton X-100, and 1X Mammalian ProteaseArrest inhibitor cocktail (G-Biosciences, St. Louis, MO, USA). 

2. Protein Quantification.

Protein quantification is performed using the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA) or Pierce protein estimation kit (Thermo Scientific, USA)

3. SDS Page Electrophoressis.

Protein lysates (typically 10–20μg) are treated with sample loading buffer containing SDS and reducing agents and incubated at 95-degree celsius for 10 minutes. 

Run 4–12% gradient NuPAGE gels (Invitrogen, Carlsbad, CA, USA) for approximately 60 minutes at 130 V. 

4. Protein Transfer.

Protein transfer is performed using the iBLOT 2 dry transfer apparatus (Life Technologies Carlsbad, CA, USA) according to the manufacturer’s protocol. 

5. Block and Addition of Primary Antibody.

Following the transfer, nitrocellulose membranes are incubated in a 1X PBS/0.1% Tween 20 blocking buffer with 5% non-fat milk for 1 h at RT, followed by incubation in the primary antibody overnight at 4°C. 

6. Addition of Secondary Antibody

After the initial incubation, the membrane is rinsed with 1X PBS/0.1% Tween 20 and incubated with the appropriate secondary antibody (Conjugated with Green or Red Dye) for 1 h at RT.

7.0 Visualization using LI-COR 

Membrane development was carried out using either the Bio-Rad chemiluminescence detection kit or the LI-COR Biosciences Odyssey CLx Imaging System. 

For quantitation, band intensities (integrated adjusted signal) were normalized t0 proteins such as β-actin or enolase using Image Studio Lite Ver 5.2 software (LI-COR Biosciences, Lincoln, NE, USA).

Immunofluorescence Staining of Protein of Interest

1. Cells are post-fixed in 4% PFA solution containing (1:1000) Stock Hoechst dye (Invitrogen, USA) for 15-30 minutes and permeabilized using 0.1% to 0.25% Triton X. 

2.0 Cells are blocked for 30-60 min in 10% normal goat serum (Sigma-Aldrich, St-Louis, MO, USA) and incubated overnight at 4°C with primary antibody. 

3.0 Secondary antibodies conjugated with Alexa Fluorophore is added and incubated.

4.0 Images are taken on the Zeiss Axio Observer Z1 microscope (Zeiss, Germany) or a Zeiss 780 confocal microscope or equivalent
.